MDA-MB-231 and MCF-7 breast cancer cells were cultured in stable isotope labeling by amino acids in cell culture Dulbecco’s modified eagle medium flex medium (A2493901, Life Technologies) supplemented with 6-mM glucose, 0.85-mM L-arginine, 0.6-mM L-lysine, 5% fetal bovine serum, and 1% glutaMAX (35050-061, Life Technologies). Cells were detached from plates using 2% ethylenediaminetetraacetic acid in phosphate-buffered saline, counted with a hemocytometer, and then plated 16 h prior to experiment. We plated in XF24 cell culture microplates (#V7-PS, Seahorse Bioscience) for seahorse analysis and on imaging dishes (fd35-100, World Precision Instrument) for ORR measurements. An hour ahead of the seahorse experiment, cells were washed twice with 1 mL of XF base medium (#102353-100, Seahorse Bioscience) before adding XF complete medium (base sodium pyruvate) and incubated at 37°C with 0% . Standard protocols for seahorse flux assay calibration and analysis were employed. All seahorse flux data were normalized to total protein content measured by bicinchoninic acid assay (#23225, Thermo Scientific) at the end of the experiment, assuming tight correlation between the protein content and cell numbers per well. The ORR of the cancer cells was measured with a commercial Zeiss LSM510 microscope equipped with a tunable ultrafast laser source (Chameleon, Coherent Inc.). In each dish, three locations were randomly selected and imaged. The fluorescence of NADH (excited at 740 nm and collected at ) and (excited at 900 nm and collected at ) was excited and collected in the epi-direction sequentially. Two imaging dishes were prepared for each cell line. We used oligomycin, FCCP, and antimycin A/rotenone in sequence on live MCF-7 cells and studied the dynamic changes of cell metabolism by the ORR and seahorse flux analysis on individual cell lines employing the same protocol. Both ORR and n-OCR were acquired every 5 min for a total of 10 measurements in 45 min. Following baseline measurements (), oligomycin was added and three measurements were performed (, and 15). FCCP and antimycin A/rotenone were added in a similar manner, right after and at , respectively, and following completion of three ORR/n-OCR measurements. Identical image planes and constant laser power were maintained for all measurements. We used a freshly prepared fluorescein solution ( at pH 7) as a reference sample for calibrating all measurements. The image intensities were adjusted based on the fluorescein reference images and analyzed with ImageJ. The region of the cytoplasm of each cell was manually selected to calculate the ORR and compared with the n-OCR measured by flux analyzer. For statistical analysis, we included the data from 12 wells for the flux analyzer ( and three experiments for each cell line) and 25 cells for ORR.